THE JOURNAL OF Bromoxa~ CHEMISTRY Vol. 244, No. 16, Issue of August 25, PP. 4406-4412, 1969

Pkhd in U.S.A.

The Reliability of Molecular Weight Determinations by Dodecyl

Sulf ate-Polyacrylamide Gel Electrophoresis*

(Received for publication, April 1, 1969)

KLAUS WEBER AND MARY OSBORN$

From the Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

SUMMARY

Forty proteins with polypeptide chains of well characterized molecular weights have been studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate following the procedure of Shapiro, Vifiuela, and Maize1 (Biochem. Biophys. Res. Commun., 28, 815 (1967)). When the electrophoretic mobilities were plotted against the logarithm of the known polypeptide chain molecular weights, a smooth curve was obtained. The results show that the method can be used with great confidence to determine the molecular weights of polypeptide chains for a wide variety of proteins.

Determination of the molecular weights of polypeptide chains in oligomeric proteins is an important problem. The most frequently used physicochemical method is equilibrium cen- trifugation in guanidine-HCl solution. For many purposes, a procedure which is experimentally less demanding but still yields reliable molecular weights would be of great value.

Shapiro, Vifiuela, and Maize1 (I) reported that the separation of proteins by polyacrylamide electrophoresis in the presence of the anionic detergent sodium dodecyl sulfate is dependent on the molecular weights of their polypeptide chains. Their results for 11 proteins were impressive, and in spite of some scatter, they showed the probable usefulness of this procedure.

We first made use of SDS* gel electrophoresis during studies on the structure of aspartate transcarbamylase of Escherichia coli. The molecular weights we obtained for aspartate trans- carbamylase were in excellent agreement with chemical and X-ray data (2, 3), and we wondered how widely applicable and how accurate this method might be. To try to answer these questions we selected some 40 proteins with well characterized polypeptide molecular weights. When the electrophoretic mo- bilities were plotted against the logarithm of the known polypep- tide chain molecular weights, a smooth curve was obtained. The results show that SDS gel electrophoresis can be used with

* This work was supported by Grant GM 16 132-01 from the National Institutes of Health.

1 Recipient of a Postdoctoral Fellowship from the Damon Runyon Fund for Cancer Research.

1 The abbreviation used is SDS, sodium dodecyl sulfate.

great confidence for a wide variety of proteins. It appears that by this technique polypeptide molecular weights may be deter- mined with an accuracy of at least =!=lOyO,.

MATERIBLS AXD METHODS

Analytical grade NaH2P0,. Hz0 and NasHPOd. 7Hz0 were obtained from Mallinckrodt Chemical Works, New York. Gla- cial acetic acid and methano! were reagent grade chemicals. Acrylamide (for electrophoresis), methylenebisacrylamide, N,N,N’,1V’-tetramethylethylenediamine and P-mercaptoeth- anol were obtained from Eastman. Ammonium persulfate, analytical grade, was a Fisher product. Bromphenol blue and Coomassie brilliant blue (R-250) were obtained from Mann. Sodium dodecyl sulfate (95’%), obtained from J!Iatheson Cole- man and Bell, was recrystallized from ethanol. Dialysis tubing 8/32 and 23/32, obtained from Union Carbide, was cleaned by standard procedures. Distilled water was used in all experi- ments.

The proteins used were obtained from Boehringer Mannheim: L-amino acid oxidase (Crotalus terr$cus), D-amino acid oxidase (pig kidney), fumarase (pig heat), and glutamate dehydrogenase (bovine liver); from Calbiochem: cytochrome c (horse heart), enolase (rabbit muscle), glyceraldehyde phosphate dehydrogenase (rabbit muscle), ,&lactoglobulin, and pyruvate kinase (rabbit muscle); from Mann: myoglobin (horse heart), lysozyme (egg white), pepsin (hog stomach), ribonuclease (bovine pancreas), serum albumin (bovine), and subtilisin (Bacilli sub!&); from Sigma: oc-chymotrypsin (bovine pancreas), cr-chymotrypsinogen A (bovine pancreas), hemoglobin (bovine), lactate dehydrogen- ase (bovine heart), liver alcohol dehydrogenase (horse liver), and yeast alcohol dehydrogenase; from Worthington: aldolase (rab- bit muscle), carboxypeptidase A (bovine pancreas), ovalbumin (egg white), papain (papaya latex), and phosphorylase a (rabbit muscle).

Carbonic anhydrase B (human blood), creatine kinase (chicken muscle), fi-galactosidase (E. coli), y-globulin (rabbit), leucine amino peptidase (bovine eye balls), paramyosin, tropomyosin, and myosin (rabbit muscle) were gifts from Drs. Edsall, Quiocho, Weil, Pappenheimer, Carpenter, Riddiford, and Lowey. The bacteriophage coat proteins (R17 and Q/I) and aspartate trans- carbamylase (E. co%) were prepared as reported (4, 2).

Performic acid oxidation of papain, subtilisin, and ribonuclease was performed by the method of Hirs (5). Carboxymethylation of lysozyme, ribonuclease, y-globulin, aspartate transcarbnm-

4406

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ylase, tropomyosin, and paramyosin was performed using iodo- acetamide in sodium dodecyl sulfate or 8 sx guanidine-IICl (6).

l’he follo\ving procedure is based on the original descriptions by Shapiro et al. (I), by Ornstein (i’), and Davis (8).

Preparation 0J Protein Solutions

The lxotcins were incubated at 37” for 2 hours in 0.01 M sodium phosphate buffer, $1 7.0, 1 y0 in SDS, and 1% in ,8mercapto- ethanol. Tropomyosin, paranlyosin, and myosin were dis- sol\-cd in this buffer in the prcsencc of 8 M urea. The protein concentration ~7~s nornlall~- betlvccn 0.2 and 0.6 mg l)cr ml. &1fter incubation the protc,in solutions IT-ere dialyzed for ,xveral hours at room temperature against 500 ml of 0.01 M sodium phosphntc buffer, $1 7.0, containing 0.1% SDS and 0.1% P-merc:il~toc:tb:I~lol. In most casts the dialysis step may be omitted and the protein dissolved directly in dialysis buffer.

Preparation of Gels

Gel buffer contained 7.8 g iY:rI-I21’0,,.H20, 38.6 g of Ns~IIl’O~~ 7H20, 2 g of SDS per liter. For the lO$G acrylamide solution, 22.2 g of acrylamide and 0.6 g of r~~ethylcnebisncrylamide were dissolved in water t,o give 100 ml of solution. Insoluble material was removed by filtration through Whatmun No. 1 filter paper. The solution was kept nt 4” in a dark bottle. Gels with in- creased and decreased cross-linker contained twice and half the concentration of cross-linker, respcctircly.

The glass gcll tubes were 10 cm long with an inner diameter of 6 111111. Before use the)- were soaked in cleaning solution, rinsed, and oreli-dried. For a typical run of 12 gels, 15 ml of gel buffer were deacrated and rnised with 13.5 ml of acrylamide solution. After further deaeration, 1.5 ml of freshly made ammonium persulfate solution (15 mg per ml) and 0.045 1111 of N, N ,N’ ,h”- tetrameth?-lethJ-leaecliamille were added. After mixing, each tube ~\-as filled with 2 ml of the solution. Before the gel hard- ened a few drops of water were layered on top of the gel solution. After 10 to 20 min an interface could be seen indicating that the gel hat1 solidified. Gels n-ith normal amount of cross-linker remain clear, those with doubled cross-linker turn opaque. *Just before use t,he mater layer was sucked off, and the tubes were placed in the clectrophoresis apparatus.

Preparation of S’amples

For each gel, 3 ~1 of tracking dye (O.O5c;/, 1)romphenol blue in \I-ater), 1 drop of glycerol, 5 ~1 of Incrcaptoeth:uiiol, and 50 ~1 of dialysis buffer ~7-a~ nlisecl in a sn~rll test tube. Then 10 to 50 ~1 of the protein solution were added. After mixing, the solu- tions were applied on the gels. Gel buffer, diluted 1 :I with I\-atcr, K:IS carefully layered on top of each sample to fill t,he tubes. The tTx-o compartments of the electrophoresis apparatus x7-erc filled with gel buffer, diluted 1:l with water. Electro- phoresis XI’ performed at a constant current of 8 ma per gel with the positive electrode in the loTIer chamber. Under these conditions the marker dye morcd three-quarters through the gel in approximately 4 hours. The time taken to run the gel may be decreased by decreasing t,he molarity of the gel buffer.2

After elect-rophorcsis, the gels xx’rc removed from the tubes by squirting lvater from n syringe b&J\-een gel and glass wall and by using a pipette bulb to exert pressure. The length of the gel and the distance moved by the dye were measured.

2 A. Burgess, personal communication.

Staining and &staining-The gels were placed in small tubes filled with st,aining solution prepared by dissolving 1.25 g of Coomassie brilliant blue in a mixture of 454 ml of 50 c/ methanol and 46 ml of glacial acetic acid, and removing insoluble material by filtration through Whatman Ko. 1 filter paper. Staining was at room teml)erature. The time varied frorn 2 to 10 hours. The gels were removed from the st,aining solution, rinsed with distilled water, and placed in destaining solution (75 ml of acetic acid, 50 ml of methanol, and 875 ml of water) for a minimurn of 30 min. The gels were then further destained electrophoretically for 2 hours in a gel clectrophoresis apparatus using destaining solution. The length of the gels after destaining and the positions of the blue protein zones T\-cre recorded. The gels were stored in 7.5y0 acetic acid solution.

The gels swell some 5% in t,he acidic solution used for staining and destaining. Gels with lower amount of cross-linker show more swelling. Therefore the calculation of the mobilit,y has to include the length of the gel before and after staining as well as the mobility of the protein and of the marker dye. Assuming even sxelling of the gels, the mobility was calculated as

Mobility = distance of protein migration

length after destaining

x length before staining

distance of dye migration

The mobilities were plotted against the known molecular weights expressed on a semi-logarithmic scale.

Time Required for Molecular Weight Determination- It is possible to obtain the molecular weight of a particular protein within a day: preparation of gels and samples, 2 hours; elcctro- phoresis, 3 to 4 hours; removing t’he gels, + hour; staining, 2 hours; destaining, 2 to 3 hours.

Amount 0J” Protein

Usually 0.01 mg of protein was applied per gel. The amount could be lon-ered if the gel was stained for a longer period. If 0.1 mg was applied although the trailing edge of the band \vas diffuse, the leading edge IT-as still very sharp and molecular weights could still be found very accurately.

E&ion of Proteins jrom Gels

On each of two gels, 0.1 mg or more was run. One gel was st.ored wrapped in Saran 1Vrap at 4”. The other gel was stained and destained to localize the protein band. The corresponding volume of the first gel was cut inlo smaller pieces. The gel material was suspended in a small amount of 0.1 o/o SDS solution and kept for several hours at 37”. The solution was withdrawn and a second elution was performed. The combined cluents were lyophilized in a conical centrifuge tube. Distilled mater was added to obtain a 1 y0 SDS solution (usually about 50 to 100 ~1). Then nine parts of ice-cold acetone were added for one part of solution. The protein precipitated and could be cen- trifuged off, whereas the detergent stayed in solution. The precipitate could be transferred to a hydrolysis tube by dissolving it in 5 7. ‘piperidine solution or in 12 N HCl.

RESULTS

Two procedures were used to examine the relationship be- tween electrophoretic mobilities and molecular weights of various proteins. Either several SDS-denatured proteins were mixed

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4408 Molecular Weights by Gel Ebctrophoresis Vol. 244, No. 16

and run together on one gel or the proteins were run in a parallel manner on different gels. Identical results were obt.ained by both methods.

Fig. 1, A, B, and C, shows examples of the separation of several different polypeptide chains on a single gel. Remarkably good separation is obtained for the polypeptide chains of cat.alase, fumarase, aldolase, glyceraldehyde phosphate dehydrogenase, carbonic anhydrase, and myoglobin (Fig. L/l). The identity of each band was established by running each protein on a different gel. In each case a single protein band with an electrophoretic mobility corresponding to the one assigned to it in the mixture was observed. Fig. 1B shows a similar mixture which was run on a different occasion, but without myoglobiu. In Fig. IC t.he same proteins were run as in Fig. IA, but substituting liver alcohol dehydrogenase for aldolase. Again, separation of all six polypeptide chains was observed. The control gel for liver alcohol dehydrogenase indicated only one band with a mobilit? corresponding to that assigned to it in the mixture. The molec- ular weights for the different polypeptide chains are taken from Table I, in which the proteins we studied by SDS gel electro- phoresis are listed. The pictures illust’rate the dependence of the mobility on the logarithm of the molecular weight of the polypeptide chains (1). The separation in the molecular weight range of 30,000 to 60,000 is excellent. It can be improved further either by increasing the length of the gel or by decreasing the amount of protein applied.

As an example of the method in which each protein is run on a

different gel, Fig. 2 illustrates the determination of the molecular

FIG. 1. Separation of the polypeptide chains of different proteins in gels with the normal amount of cross-linker. The proteins are listed from lop to bottom, and the molecular weights given for the different polypeptide chains are taken from Table I. A, catalase (60,000), fumarase (49,000), aldolase (40,000), glycer- aldehyde phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), and myoglobin (17,200); B, same as A, but omitting the myoglobin. This gel was run on a different occasion; C, catalase, fumarase, liver alcohol dehydrogenase (41,000), glyceraldehyde phosphate dehydrogenase, carbonic anhydrase, and myoglobin.

TABLE I Proteins studied bv SDS-electrophoresis

The table lists molecular weights of the polypeptide chains taken from the literature. Proteins which under native condi-

tions exist as oligomers are indicated by an asterisk.

Protein

Myosin*. .................... p-Galactosidase*. ............

Paramyosin ................. Phosphorylase a*. .......... Serum albumin. .............

L-Amino acid oxidase. ...... Cat,alase*, .................. Pyruvate kinase*. ........ : Glutamate dehydrogenase*. .. Leucine amino peptidase ..... r-Globulin, H chain*. .......

Fumarase*. .................. &albumin. ................ Alcohol dehydrogenase (liver) Enolase* .................... Aldol ase*. ...................

Creat,ine kinase*. ............ D-Amino acid oxidase*. ......

Alcohol dehydrogenase (yeast) * ..................

Glyceraldehyde phosphate dehgdrogenase*. ...........

Tropomyosin* ............... Lactat,e dehydrogenase*. ..... Pepsin .......................

Aspartate transcarbamylase, C chain*. .................

Carboxypeptidase A. ........

Carbonic anhydrase .......... Subt.ilisin”. .................. r-Globulin, L chain.*. ....... Chymotrypsinogen. .........

Trypsin .................... Papain (carboxymethyl) ..... p-Lactoglobulin*. ............ Myoglobin ..................

Aspartate transcarbamylase, R chain*. ................

Hemoglobin*. ..............

&p coat protein .............. Lysozyme .................. R17 coat protein ............. Ribonuclease ................

Cytochrome c ............... Chymotrypsin*, 2 chains .....

*

Mel wt of polypeptide chain Reference

. - 220,000 9, 10, 11 130,000 12 100,000 13 94,000 12, 14 68,000 15 F3 ) 000 1G 60,000 17, 18 57,000 19 53,000 12, 20, 21 53 ) 000 Q

50,000 22

49,000 23 43,000 24 41,000 24 2jb

41,000 21; 26 40,000 24, 27, 28 40,000 29

37,000 30

37,000 31c

3G, 000 32, 33 36,000 13, 34 36,000 24 35,000 35

34,000 34, GO0 2!1,000

27,600 23,500 25,700

23,300 23,000 15,400 *

17,200

17,000

15,500 15,000 14,300

13,750 13,700 11,700

11,000 and 13,000

2, 3 36

37

38 22

c e

e. f

15 e

2, 3 c

400 c e 8 e

c

0 K. Weber, unpublished results. b JORNVALL, H. AND HARRIS, J. I., Abstracts of the Federation of

EuropeaTL Bigchemical Sciences, Praha, 1968, abstract 759.

c BUTLER, P. J. G. AND HARRIS, J. I., Abstracts of the Federation of European Biochemical Sciences, Praha, 1968, abstract 741.

d After performic acid oxidation. 8 Calculated from the amino acid sequences given in Dayhoff

and Eck (39). f Corrected according to the X-ray structure (J. Drenth, per-

sonal communication).

0 W. Konigsberg, personal communication.

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IO I I I 9 8 7 -1

p: 6 c

I I 0.2 0.4 0.6 0.8 1.0

Mobility

FIG. 2. 1)elerminatiou of the molecular weight of the poly- peptidc elmin of u-amino acid oxidase from a set of 12 individual stnntl:trcl gels. The six marker proteins rlsed were glrllamic acid dehydrogcnase, flmmrase, aldolase, glyceraldehydc phosphate d&J-tlrogcnase, trypsill, and m\-oglohin. All proteins were run on dllplic:~tc gels except glu~:~m~c dehydrogenase and myoglobin. The C~~‘WXU indicates the mobili(y of D-amino acid oxidase from t\vo dil’l’crellt gels (0.398; 0.102). The molecular weights of the marker proteins are taken from Table I. The estrapolated valae for I)-:mlilro acid oxidase is 37,000.

weipjit of namiiio ncid osidasc. A@1 the clectrophorctic mobili!ics for marker polypcl~~itlc chains are plotted xpaiust the log of their niolccular w$hts. From the elcctrophorctic Inobility of w:rlliiuo acid osidnse, a nlolccular weight of 37,000 ix found.

‘I’hc rclxoducibility of the I;>-atcm can be illustlatcd bh- the follo\vilig cq)eriment. ~\sJKLrt:ltc tlalls:c:r~banl~-l:lsc Iron1 I$. coli

KRS r1111 in :I parallel I~:III~CI’ on 12 individual gels. Two pro&in bands \VCW: obtained which corrcsl)ond to the two different polylwl)tidc chains of this cnzync (2). The clcc’trol)horctic mobilitics I\-crc 0.44 to 0.16 for the C Band and 0.73 to 0.76 for the It 1i:md. The arcwgc mobilitics calculated from these 12 gels \v(lrc 0.435 + 3c, :rlld 0.71~5 i 3L;$, rtsl)ccti\-cly. From these rr~ults the rel)rotlllci})ilit?- a1q)cars better than 5:;. Ilo\\-- PT-cr, 111~ absolute ~~lucs of the rnobilit~~ of the ,samc pal)-l)cl)tide chain l’u11 on selxuxte occxsions sometimes sholvs :L dcri:rtion of 5 t,o 10“;. This slight de\-iation scc~ns to occur with IIWV b:ltches of l)ol!-ac.r!-larriiclc. Howcvcr, in ~11 GAYS, lhe plot of the mo- bilitiw for :L given set of stund:rrds gives thr sanic value for the molccul:~r weight of a lwticular lxotein.

The itlcntity of a particular lxotcin band has brcn rcl~atedlg established by the elution l~roccdure given in the l)rcriolw sec- tioll, l’olloxcd by amino wid analysis. Greater than 75y0 recovcry may be obtained by this method. The rncthotl :rlloc~-s easy sclwation and recovery 01 small quantities of lxotcins that are sufticicnlly different in molwular x-eight.

The lxoc*edure may bc :rd:~ptcd to study different molecular weight ranges. This GH~ be sccn iu Fig. 3. Six different polg- l)cl)t idc clGns were run wing 107; acrylamide solution, but changing the amount of IIlcth?-lcnebisacr~l~lrllidc. ,‘L drastic change in the mobilitics occurs. For example, the mobility of

IO 9 8

7

glyceraldehyde phosphate dehydrogenase

02 0.4 06 08 IO

Mobility

FIG. 3. I<:lecirophoretic mobility as a frmction of the amormt of cross-linker. The mobility is shown for the polypcptide chains of frmuxase, aldolasc, glyceraldehyde phosphate dchydrogelrase, trypsiil, myoglobin, and R17 coat prot,eirr using the normal amomlt of cross-linker (n~iclclle CICTW), 0.5 (righl cr~we), and 2

times the amount (rc:Jt cuwc).

funl:w~c with a molecular wcighl of 50,000 changes from 0.28 to 0.63 if the alnount of cross-lillkcr is reduced from the 11orma1 amount to half of this \-alue. This suggests that gels with half the normal amounts of cross-lillkcr would be us;c~ful to stud? proteins over :L wide rnngc of molecular weights (we bclon-).

The results of our meaxwemcnts on close to -10 tlitfrwnt 1x0. teins arc ,+hown in Figs. 3 and 5 where the rnobilitics arc l~lottccl agaiwt the log of the n~olccular weight &ken from Table 1. Each protein has been run on at least four different 0ccGons. Fig. A shons wsults for 37 different l)olylwptidc chains ill the ~~~oleculnr n-eight r:iilge from 10,000 to 70,000. hi no c:l>e has :L wrious de\-iation from the niolccular wciglits gi\.en ii1 ‘l’al)lc I been showll. The maximum deviation from the prctlic+xl \-aluw in this ra~~gc i,* Iws thau 105, an d [or nlost, lxoteius the agreement is bcttcr. This accuracy is crlw;tcd bccausc, as sho\vll ill Fig. t, l)olyl)cl)tidc chains differing by oldy 105~1 in wcigllt wn be separated on the same gel.

Fig. 5 almwh the results for 10 diffcrpnt l)olylwl)titlc chains in the higllc~ nlolrculx weiglil wlrgc from 50,000 to 200,000. Gels

with h:l!f the ~~orn~~l an~oullt of c-rash-linker \YPI’C 1~x1 ill thc,+e cspwitrwlit-. 111 spite of the hylwbolic curve obtaiiwtl (xc also Sh:rl)iro el rri. (1) and Fig. 3), the rnobilitics loilo~\- the, kno\~n n~olc~ul:tr \vcights. ALlthougll ~cwcr standards arc avuilablc for this ixilgc, an accuracy of l IO’L, still seems possible.

Our rcwltx shorv that, mwuingful molecular weights c:ln be obtained by 81)s gel elcctrol)horcsis for the three rnusclc pro-

teins trolmtnyosin, paraniyosin, and inyosin. TfTe wvcrc interested in these 1)roteins because they arc known to have a high helical colltcllt. Tropomyosin yields a molecular wight of 36,000 in good :qgwment with the ~alucs given in the literature (13, 3-1). Using vccy low protciii concentrations we found that the 1)rolciri actually did not give a single band, but a doublet thal LV;LS hnrdl~ separated. At the present, it is unclear whether this is an artifact with the lxcparation we hnvc available or mhcthcr the poly- peptide chains of tropomyosiii arc not identical. I’aramyosin yielded the expected pol~pcl~tide chain of molecular weight 100,000 (13). For myosin KC obtained a polypeptitlc chain

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Molecular Weights by Gel Rlectrophoresis Vol. 244, No. 16

0.2 0.4 0.6 0.8 1.0

Mobility FIG. 4. Comparison of the molecular weights of 37 different

polypeptide chains in the molecular weight range from 11,000 to 70,OOOwith their elcctrophoretic mobilities on gels with the normal amount of cross-linker. The references to the moleclllar weights are given in Table I.

with a molecular weight close to 200,000, in agreement with the reported values for this protein (9, 10). Howcvcr, we also de- tected three minor components of lower molecular weight,s (16;OOO to 23,000). At this time, we don’t know if these com- ponents are inlpurities in the preparation KC had available or if they are inherent components of myosin. The latter assumption is supported by recent results obtained in different laboratories.3

In a few rases we studied the proteins after performic acid oxidation or carbox~meth~l:~tiol~. The electrophoretic mobil- ities of papain and ribonuclease aft’er performic acid oxidation were identical with the ones obtained before. Ko differences were seen after carbosymebhylation for lysozymc, aspartate transcarballl3-lase, tropomyosin, and paramyosin. This indi- cates that the P-mercaptoethanol prevents the formation of interchain cystine bonds. This assumption is supported by the fact that we did not find dimers of polypcptide chains. The possibility that some rare protein will not be delnatured by the SDS treatment in the presence of P-mercaptocthanol suggests that a control should be performed using an alkylated or oxidized derivative.

3 S. Lowey, personal communication.

myosin

/.?-goloctosidase

phosphorylase a

serum albumin L-amino acid oxidase

glutamic dehydrogenase

0.2 0.4 0.6 0.8 1.0

Mobility FIG. 5. Comparison of the molecular weights of different

polypeptide chains (see Table I) in the molec\llar weight range from 40,000 to 200,000 with their electrophoretic mobilities on gels with half the amormt of cross-linker.

DISCUSSION

Separation of native proleins on polyacrylamidc gels was shown by Ornstein (7) and T>avis (8) to be dependent not only on the charge but very strongly on t,he size of the molecules. The binding of dodecyl sulfate ions to proteins has been shown for several protein molecules (see Tanford (41) for :I recent re- view), and was assumed to be the basis of the separation of the denatured proteins upon SDS electrophoresis on l)olyacrylamide (1). I f so, one must assume that the individual charge pattern of each protein is totally changed by the binding of SDS anioix, rendering all molecules negatively charged. At pre;ent it is

difficult to see why proteins that differ widely in amino acid com-

position and isoelectric points should all follow the general pat- tern. It is possible that the sieving effect, which is an cxpo- nential function, overcomes the charge effect which may be of minor importance. Because of these theoretical difficulties we will discuss the results only from a practical point of view.

Two questions were raised during this study. How reproduc- ible are the results obtained by SDS gel electrophoresis and how reliable are the molecular weights obtained? The results pre- sented above quite clearly show that a high degree of reproduci- bility in the determination of the electrophorctic mobility can be obtained whether the proteins are run on different gels or on the same gel.

We have shown that close to 40 different prot,eins have clec- trophoretic mobilitics which are independent of the isoelectric point and the amino acid composition and seem governed solely by the molecular weights of their polypeptide chains. Since the major&y of the polypeptidc chains used are either characterized by molecular weights known from amino acid sequence analysis,

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Issue of August 25, 1969 K. Weber and M. Osborn 4411

or X-ray crystallography, or by very careful studies in guanidine- HCl with similar results obtained in different laboratories, we feel confident that this study is a very stringent test for the technique of Shapiro et al. (1). All the proteins studied yielded molecular weight’s within the experimental error of the known values. We have not studied proteins containing large amounts of carbohydrate or lipid material. It is possible that such pro- teins, because of their large nonprotein part, will behave dif- ferently on gel electrophoresis. It is, however, noteworthy that SDS gel electrophoresis yields not only excellent results for pro- teins, which are globular in the native state, but also for the highly helical, rod-shaped molecules, myosin, paramyosin, and tropomyosin. For all three proteins we obtained polypeptide chains with molecular weights in agreement with the values from the literature (see “Results”).

With these results one is inclined to assume that the technique of Shapiro et al. (1) yields molecular weights with an accuracy of better than +lO% for polypeptide chains with molecular weights between 15,000 and 100,000. This range can be covered with numerous commercially available proteins as standards. The difficulty with higher molecular weights is probably only the fact. that fewer markers are available for the range 90,000 to 200,000. Polypeptide chain markers commercially available for this range are phosphorylase with a molecular weight of 94,000 (12, 14) and thyroglobulin with a molecular weight of 160,000 (42). The preparation of P-galactosidase from E. coli can be easily accomplished (43) and yields a marker band with 135,000 molecular weight (12). Myosin and paramyosin can usually be obtained from the numerous laboratories working on these pro- teins and yield additional markers with molecular weights of 200,000 and 100,000. With enough markers an accuracy of about 10% in the determination of the molecular weight of an unknown protein falling in this molecular weight range may be possible.

Four examples will be used to illustrate these points further. Phosphorylase a gave a single polypeptide chain of molecular weight 94,000 without any indication of heterogeneity. This is in agreement with the results of Ullmann et al. (12), using the approach to equilibrium centrifugation in the presence of 6 M

guanidine-HCl. Seery, Fischer, and Teller (14) found a similar value by equilibrium centrifugation in the same solvent only after accounting for some heterogeneity and preferential hydra- tion. The latter, however, is an unlikely phenomenon in view of the results obtained by Kirby Hade and Tanford (44), Reithel and Sakura (45) and Ullmann et al. (12).

A further sample is liver alcohol dehydrogenase. The tech- nique of Shapiro et al. (I) yields a value of 40,000 in agreement with results obtained by osmotic pressure measurements in guanidine-HCl (24), X-ray analysis (25), and preliminary se- quence analysis4, but contrary t,o prior sedimentation analysis studies (46).

The separat,ion of the polypeptide chains of fumarase, aldolase, and glyceraldehyde phosphate dehydrogenase on one gel is very striking. The molecular weights of these polypeptide chains are today well established. Aldolase was for some time assumed to have a molecular weight of 50,000. This value was mainly based on preferential hydration in guanidine-HCl (47). How- ever by using glyceraldehyde phosphate dehydrogenase with a known amino acid sequence as one marker and fumarase, which

4 J~~RNVALL, H., AND HARRIS, J. I., Abstracts of the Federation of European Biochemical Sciences, Praha, 1968, abstract 759.

is well characterized, as the other, it can be concluded that the molecular weight of the aldolase polypeptide chain is 40,000. Meanwhile, this is the value accepted for careful measurements by ultracentrifugation (27) and osmotic pressure (24) in guani- dine-HCl, as well as from chemical studies (28).

Conflicting values for the size of the polypeptide chain of n-amino acid oxidase are found in the literature and have been summarized recently by Henn and Ackers (30). They showed a value of 35,000 to 40,000 by gel filtration. The SDS electro- phoresis yields 37,000. From all the evidence presented for the other proteins we think that the two values indicating 37,000 are much better than the one of 50,000 reported by Fonda and Anderson (48). Their value is based mainly on fingerprint analysis and a gel filtration study, which is less extensive than the one by Henn and Ackers (30).

Since the method appears to yield values in agreement with the best current estimat,es for all the proteins studied, it seems fair to compare SDS gel electrophoresis with other methods. The excellent resolving power of the gels over a wide range of mo- lecular weights has been illustrated. In this respect the method is much superior to gel filtration patterns on Sephadex in a de- naturing solvent like guanidine-HCl (49). The good resolution and the fact that an estimate of the molecular weight can be obtained within a day, together with the small amount of pro- tein needed, makes the method strongly competitive with others commonly employed. The theoretically fully developed meth- ods of osmotic pressure and sedimentation equilibrium in 6 M guanidine-HCl are still superior. However, osmotic pressure measurements suffer from the large amount of protein needed and the fact that extremely accurate protein determination is necessary. Both limitations do not apply for sedimentation equilibrium using interference optics. However this method is very demanding experimentally if good results are to be obtained. But even in the optimal case the calculation depends on the value chosen for the partial specific volume V. An uncertainty of 0.02 in this value introduces a deviation of up to lo’%, even if all the centrifuge measurements are performed well. Also, in some cases, in spite of the fact that there is good evidence for no major change in ti in guanidine-HCl (12, 44, 45), the assump- tion of preferential hydration is still quite often used (14, 47) to account for deviations.

It is by no means intended to assume that the accuracy of SDS gel electrophoresis will be comparable with the well de- veloped physicochemical methods. Also, certain theoretical aspects of SDS gel electrophoresis are still not clearly under- stood. In spite of these limitations, however, the ease with which the method can be applied, together with the results presented above, encourage its wider use, especially in case of conflicting data obtained by other methods.

Acknowledgments-We thank Elizabet’h Bloomquist for excel- lent and skillful assistance with many of these experiments. Discussions with Dr. R. Burgess, Dr. G. Guidotti, and Ann Burgess were most helpful.

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Klaus Weber and Mary OsbornSulfate-Polyacrylamide Gel Electrophoresis

The Reliability of Molecular Weight Determinations by Dodecyl

1969, 244:4406-4412.J. Biol. Chem. 

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